Use of melatonin as an inhibitor of apoptotic process for cryopreservation of zebrafish (Danio rerio) embryos / Uso da melatonina como inibidor do processo apoptotico para a criopreservação de embriões de zebrafish (Danio rerio)

This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.

Este estudo investigou o uso da melatonina para conter os efeitos da apoptose em embriões vitrificados de zebrafish (D. rerio). Embriões descorionados no estágio de 22-24 somitos foram divididos (n = 60 / tratamento) em tratamento não vitrificado (Grupo Controle, melatonina 0 M) e tratamentos vitrificados com 0 M (T1), 1 µM (T2) e 1 mM de melatonina (T3). Para os tratamentos vitrificados, utilizou-se uma solução à base de metanol/propilenoglicol e os embriões foram armazenados em -196 °C por uma semana. Após o descongelamento, foram realizadas análises de taxa de sobrevivência, microscopia eletrônica de varredura, expressão dos genes anti (bcl-2) e pró-apoptóticos (bax/caspase-3), formação de espécies reativas de oxigênio (EROS) e análises de fragmentação de DNA. Não foram obtidos embriões vivos a partir dos tratamentos vitrificados, observando uma rápida degeneração imediatamente após o descongelamento, com ruptura da camada vitelina e vazamento de seu conteúdo, seguida de quebra das células epiteliais e melanização do tecido. Em relação ao processo apoptótico. T3 apresentou expressão gênica relativa alta para os três genes (P 0,05). A inclusão de 1 µM de melatonina na solução de vitrificação, contrariou os efeitos do processo apoptótico em embriões pós-descongelamento, sugerindo sua utilidade na criopreservação de embriões de peixes.

Use of melatonin as an inhibitor of apoptotic process for cryopreservation of zebrafish (Danio rerio) embryos

Abstract This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P 0.05) furthermore, T2 had similar expression of pro-apoptotic genes to CG (P 0.05). ROS formation revealed that CG presented lower percentage of embryo surface area affected (3.80 ± 0.40%) (P 0.05), in contrast, no differences were found among the other groups. T1 was most significantly (P 0.05) damaged by DNA fragmentation. The vitrified groups with melatonin had similar damage levels of CG (P > 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.

Resumo Este estudo investigou o uso da melatonina para conter os efeitos da apoptose em embriões vitrificados de zebrafish (D. rerio). Embriões descorionados no estágio de 22-24 somitos foram divididos (n = 60 / tratamento) em tratamento não vitrificado (Grupo Controle, melatonina 0 M) e tratamentos vitrificados com 0 M (T1), 1 µM (T2) e 1 mM de melatonina (T3). Para os tratamentos vitrificados, utilizou-se uma solução à base de metanol/propilenoglicol e os embriões foram armazenados em -196 °C por uma semana. Após o descongelamento, foram realizadas análises de taxa de sobrevivência, microscopia eletrônica de varredura, expressão dos genes anti (bcl-2) e pró-apoptóticos (bax/caspase-3), formação de espécies reativas de oxigênio (EROS) e análises de fragmentação de DNA. Não foram obtidos embriões vivos a partir dos tratamentos vitrificados, observando uma rápida degeneração imediatamente após o descongelamento, com ruptura da camada vitelina e vazamento de seu conteúdo, seguida de quebra das células epiteliais e melanização do tecido. Em relação ao processo apoptótico. T3 apresentou expressão gênica relativa alta para os três genes (P 0,05), além disso, T2 apresentou expressão semelhante as dos genes pró-apoptóticos de GC (P 0,05). A formação de EROS revelou que GC apresentou menor percentual de área de superfície embrionária afetada (3,80 ± 0,40%) (P 0,05), ao contrário, não foram encontradas diferenças entre os outros grupos. T1 foi mais significativamente (P 0,05) danificado pela fragmentação do DNA. Os grupos vitrificados com melatonina apresentaram níveis de dano semelhantes ao do GC (P> 0,05). A inclusão de 1 µM de melatonina na solução de vitrificação, contrariou os efeitos do processo apoptótico em embriões pós-descongelamento, sugerindo sua utilidade na criopreservação de embriões de peixes.

Use of melatonin as an inhibitor of apoptotic process for cryopreservation of zebrafish (Danio rerio) embryos / Uso da melatonina como inibidor do processo apoptotico para a criopreservação de embriões de zebrafish (Danio rerio)

This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P < 0.05) furthermore, T2 had similar expression of pro-apoptotic genes to CG (P < 0.05). ROS formation revealed that CG presented lower percentage of embryo surface area affected (3.80 ± 0.40%) (P < 0.05), in contrast, no differences were found among the other groups. T1 was most significantly (P < 0.05) damaged by DNA fragmentation. The vitrified groups with melatonin had similar damage levels of CG (P > 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.

Este estudo investigou o uso da melatonina para conter os efeitos da apoptose em embriões vitrificados de zebrafish (D. rerio). Embriões descorionados no estágio de 22-24 somitos foram divididos (n = 60 / tratamento) em tratamento não vitrificado (Grupo Controle, melatonina 0 M) e tratamentos vitrificados com 0 M (T1), 1 µM (T2) e 1 mM de melatonina (T3). Para os tratamentos vitrificados, utilizou-se uma solução à base de metanol/propilenoglicol e os embriões foram armazenados em -196 °C por uma semana. Após o descongelamento, foram realizadas análises de taxa de sobrevivência, microscopia eletrônica de varredura, expressão dos genes anti (bcl-2) e pró-apoptóticos (bax/caspase-3), formação de espécies reativas de oxigênio (EROS) e análises de fragmentação de DNA. Não foram obtidos embriões vivos a partir dos tratamentos vitrificados, observando uma rápida degeneração imediatamente após o descongelamento, com ruptura da camada vitelina e vazamento de seu conteúdo, seguida de quebra das células epiteliais e melanização do tecido. Em relação ao processo apoptótico. T3 apresentou expressão gênica relativa alta para os três genes (P <0,05), além disso, T2 apresentou expressão semelhante as dos genes pró-apoptóticos de GC (P <0,05). A formação de EROS revelou que GC apresentou menor percentual de área de superfície embrionária afetada (3,80 ± 0,40%) (P <0,05), ao contrário, não foram encontradas diferenças entre os outros grupos. T1 foi mais significativamente (P <0,05) danificado pela fragmentação do DNA. Os grupos vitrificados com melatonina apresentaram níveis de dano semelhantes ao do GC (P> 0,05). A inclusão de 1 µM de melatonina na solução de vitrificação, contrariou os efeitos do processo apoptótico em embriões pós-descongelamento, sugerindo sua utilidade na criopreservação de embriões de peixes.

Use of melatonin as an inhibitor of apoptotic process for cryopreservation of zebrafish (Danio rerio) embryos.

This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P < 0.05) furthermore, T2 had similar expression of pro-apoptotic genes to CG (P < 0.05). ROS formation revealed that CG presented lower percentage of embryo surface area affected (3.80 ± 0.40%) (P < 0.05), in contrast, no differences were found among the other groups. T1 was most significantly (P < 0.05) damaged by DNA fragmentation. The vitrified groups with melatonin had similar damage levels of CG (P > 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.

Effects of bisphenol A and fadrozole exposures on cyp19a1 expression in the Murray rainbowfish, Melanotaenia fluviatilis.

Several endocrine-disrupting chemicals (EDCs) have been attributed to the alteration of reproduction in fish through disrupting endogenous sex steroidogenic pathways including aromatisation of androgens to oestrogen by CYP19 aromatase. Here we investigate this hypothesis in adult male and female Melanotaenia fluviatilis by examining the mRNA expression of cyp19a1 isoforms after exposure for ≤96 h to two EDCs with contrasting modes of action: one a weak oestrogen mimic, bisphenol A [BPA (100 or 500 µg/L)], and the other a nonsteroidal aromatase inhibitor, fadrozole [FAD (10 or 50 µg/L)]. The results suggest that BPA did not affect cyp19a1a expression significantly at both concentrations, whereas 50 µg/L of FAD significantly upregulated its expression in ovary. In contrast, BPA exposures increased expression of cyp19a1b in brain of both males and females, whilst FAD had contrasting effects in brain: It increased in males but decreased in females. Similar contrasting responses of cyp19a1b were induced by BPA in gonads: upregulation in ovary and downregulation in testis. FAD did not have a significant effect on gonadal expression of cyp19a1b. Collectively, the results suggest that BPA and FAD can disrupt cyp19a1b activity more readily than can cyp19a1a, albeit with contrasting effects in either a tissue- or sex-specific context that is conceivably consistent with their (BPA and FAD) opposing modes of action. Enhanced spatial and temporal sensitivity of cyp19a1b compared with cyp19a1a suggests that brain sex of fish is more susceptible to disruption by environmental pollutants such as BPA and FAD. Therefore, we propose that the response of cyp19a1b in brain tissue of M. fluviatilis is a more suitable indicator of oestrogenic pollution in the aquatic environment.

Exposure to estrogenic chemicals induces ectopic expression of vtg in the testis of rainbowfish, Melanotaenia fluviatilis.

Vitellogenin (Vtg) is the major egg-yolk precursor protein in oviparous organisms normally synthesised only in mature females. In males and juveniles, the vtg gene, although present, is silent, but its hepatic expression may be activated by xenoestrogens. Surprisingly, its induction and potential consequences in non-hepatic tissues remain unexplored. Here we test the hepatic and testicular response of vtg expression in adult male rainbowfish Melanotaenia fluviatilis exposed to either 1, 3, 5 µg/L 17ß-estradiol or 100, 500 µg/L 4-n-nonylphenol for 24-96 h. Significant increase in the expression level of vtg mRNA in the liver and testes of exposed males was observed. The early (24 h), sensitive and reliable detection of the vtg induction using qPCR demonstrates the assay's robustness to monitor xenobiotic exposure particularly in smaller fish like rainbowfish, an emerging indicator species. Whilst, the ectopic induction of vtg mRNA in testes suggests a more complex Vtg pathway.

Effects of exposure to oestrogenic compounds on aromatase gene expression are gender dependent in the rainbowfish, Melanotaenia fluviatilis.

This study investigated the influence of two endocrine disrupting chemicals (EDCs)-an exogenous oestrogen 17ß-estradiol (E2) and the oestrogen mimic 4-n-nonylphenol (NP) on the expression of aromatase transcripts in both sexes of adult Murray river rainbowfish. Reproductively active mature fish were exposed to 1, 3, and 5 µg/L E2 or 100 and 500µg/L NP for 24, 48, 72 and 96 h. The results show a significant reduction in the expression of cyp19a1a isoform in ovarian tissues with complete inhibition at the higher concentrations (3 and 5 µg/L E2; 500µg/L NP between 24 and 72 h) and at all concentrations after 96 h. There was no expression of the cyp19a1a isoform in female brain, male brain or testes in any treatment. E2 significantly increased expression of cyp19a1b in female brain except at 5 µg/L after 24h exposure. In male brain tissue E2 exposure decreased cyp19a1b expression except at 1 and 5 µg/L at 24h. NP significantly upregulated cyp19a1b in the female brain (except with 500 µg/L at 72 h) and in testes tissues. NP downregulated expression of cyp19a1b in the male brain tissue. Collectively, these observations support the hypothesis that the expression of cyp19a1b is regulated via both positive and negative feedback mechanisms, with differential modulation based on the type and concentration of the exposed oestrogens, duration of exposure, fish tissue and gender of the fish. The results also imply that exogenous oestrogens can have a disruptive effect on the steroidogenic pathway and may lead to effects on sex differentiation, sexual behaviour and reproductive cycles in this fish.

Ontogenic and sexually dimorphic expression of cyp19 isoforms in the rainbowfish, Melanotaenia fluviatilis (Castelnau 1878).

To investigate the role of cytochrome P450 aromatase, we isolated cyp19 isoforms in the Murray River rainbowfish, M. fluviatilis. The cloned cDNA for cyp19a1a and cyp19a1b had an open reading frame (ORF) of 492 and 499 amino acid residues, with shared identity of up to 83% and 87% with the corresponding homologues of other teleosts respectively. In contrast, the cyp19a1a and cyp19a1b of the Murray River rainbowfish had a shared identity of only 61%. Not surprisingly, the phylogenetic analysis clustered the M. fluviatilis cyp19 isoforms with the corresponding isoforms of other teleosts, suggesting a shared evolutionary ancestry of the respective isoforms. We also studied the expression of cyp19 isoforms during ontogeny and in adult fish using quantitative Real-Time PCR (qPCR). Results suggest that uniquely only cyp19a1b transcripts are maternally inherited, suggesting its role in early development and growth in the species. In contrast to reports in many teleosts, the cyp19a1a was exclusively expressed in the ovarian tissue and completely absent in other tissues examined, including testis. The cyp19a1b like in most teleosts was predominantly expressed in the brain of both males and females with low level of expression in other tissues including gonads of both sexes.

Nuclear internalization of foreign DNA by zebrafish spermatozoa and its enhancement by electroporation.

Mature sperm cells of zebrafish (Danio rerio) incubated with foreign DNA have the capacity to take up foreign DNA. Such uptake can be enhanced by electroporation. Mature spermatozoa of zebrafish were incubated and electroporated in the presence of either radiolabeled or unlabeled plasmid DNA at voltages of 500 or 1,000 or 1,500 V/cm. From the percentage of radiolabeled plasmids retained on the spermatozoa, some sperm showed an ability to spontaneously take up the plasmid DNA, and the ability was enhanced one- to twofold by electroporation. Fertilization of mature eggs with the treated sperm resulted in transmission of the plasmid DNA to the resulting offspring. Frequency of transgenic individuals, as monitored by polymerase chain reaction, increased marginally, more than doubled and nearly doubled in 500 V/cm, 1,000 V/cm, and 1,500 V/cm electroporated groups, respectively, when compared to the non-electroporated group. These results indirectly implied that electroporation enhanced the capacity of spermatozoa to take up plasmid DNA. The increased field strength, however, had a deleterious effect on the motility of the sperm, causing clumping of sperms at high voltages. Light microscopic autoradiography of treated spermatozoa was able to show that the plasmid DNA was associated with the majority of sperm but was unable to differentiate whether it was present inside the nucleus or not. Ultrastructural in situ hybridization on thin sections of zebrafish spermatozoa, however, was able to show that the exogenous DNA was internalized into the nucleus and that electroporation enhanced this internalization. The results provide direct evidence for nuclear internalization of foreign DNA by non-mammalian sperm as in mammalian sperm.

Ultrastructural in situ hybridization and autoradiographic detection of foreign DNA in zebrafish spermatozoa.

Conditions have been established which permit specific hybridization of foreign DNA to complimentary DNA in ultra thin sections of zebrafish sperm prepared for electron microscopy (EM)1 after fixation in 2% paraformaldehyde, 2.5% glutaraldehyde and embedded in spurs resin. Zebrafish sperm incubated with foreign DNA were subjected to ultrastructural in situ hybridization and autoradiographic detection to determine if the sperm could retain and internalize the foreign DNA sequences. Specific and intense signals were detected from the sperm head indicating that the zebrafish sperm are capable of internalizing foreign DNA.

Assessment of pMTL construct for detection in vivo of luciferase expression and fate of the transgene in the zebrafish, Brachydanio rerio.

Early embryos of the zebrafish Brachydanio rerio were cytoplasmically microinjected with pMTL plasmid containing firefly luciferase gene, in both linearized- and supercoiled-plasmid forms, to evaluate in vivo expression, pattern of integration and germ-line transmission of the transgene in the host fish. It was possible to detect luciferase expression in vivo, and the pattern of time-course expression was similar in both linearized- and supercoiled-plasmid injected groups. Strong luciferase activity was detected 15-20 hours after injection, coinciding with early somitogenesis. Expression was detectable in a few 1 week-old individuals but was not detectable in all adults and in F1 progeny. In vivo screening for expression of the transgene in the developing embryo using luciferase assay as a method for detecting the presence of the transgenic fish compares favourably, with PCR and Southern blot analysis (SBA). No integration of the introduced DNA into the genome of treated fish and their progeny, was detected, instead it remained in extrachromosomal form. Most of the first generation founders were mosaic. Germline transmission was observed in one individual only. A probable reason for the absence of integration in this study when compared to the varying frequencies reported earlier in the same fish is discussed.